The brassinosteroids (BRs) are a group of plant steroid hormones that help regulate many different aspects of plant growth and development. BRs are known to stimulate cell elongation and division, and are also involved in vascular system differentiation, reproduction, and stress responses (Altmann, 1998; Clouse and Sasse, 1998). Recently, it has been shown that mutants defective either in BR biosynthesis or signaling, display altered developmental phenotypes including dwarfism, reduced fertility, and abnormal vasculature (Clouse and Feldmann, 1999).
BR dwarf mutants can be divided into two classes. The first class of mutants is perturbed in BR signaling. For example, Clouse, et al., (1996) isolated a signaling mutant, brassinosteroid insensitive1 (bri1), that was resistant to exogenously applied BRs. BRI1 has been cloned and shown to encode a leucine-rich repeat receptor kinase, suggesting a role for BR perception at the cellular membrane (Li and Chory, 1997). Recently, it was demonstrated that bri1 mutants accumulate significant amounts of brassinolide (BL) and its precursors compared to wild-type controls, suggesting that perception is coupled to homeostasis of endogenous BR levels (Noguchi, et al., 1999b).
The other class of BR mutants includes a large number of dwarfs that are defective in BR biosynthesis. Plants produce BRs using sterols as precursors, and the sterol biosynthetic pathway uses mevalonic acid as a precursor to synthesize sterols, such as sitosterol, stigmasterol, and campesterol. Sterols are modified by the BR-specific pathway to produce the end product, BL, and its congeners. Thus, mutants that are defective in either the sterol or BR-specific pathway display a typical BR dwarf phenotype, and can be rescued to a wild-type phenotype by exogenous application of BRs.
The characteristic phenotype of BR dwarf mutants has been instrumental in isolating additional mutants, and their corresponding genes perturbed in the complex plant sterol biosynthesis network. dwf1 was the first mutant isolated to have this dwarf phenotype (Feldmann, et al., 1989). The dwf1 mutant is defective in C-24 reduction, and DWF1 encodes a FAD-binding oxidoreductase (Choe, et al., 1999a; Klahre, et al., 1998; Takahashi, et al., 1995). The pea lkb mutant is deficient in the same reaction as Arabidopsis dwf1 (Nomura, et al., 1999). Another sterol mutant, Arabidopsis dwf7/ste1, has been isolated and found to be defective in the Δ7 sterol C-5 desaturase gene (Gachotte, et al., 1995; 1996, Husselstein, et al., 1999, Choe, et al., 1999b).
Currently, little is known about the downstream events that occur in response to signals in the above pathways that ultimately control cell size. This is because the biochemical and cell biological processes involved have thus far been difficult to address. In addition, there is little information about the integration of regulatory signals converging at the cell from different signaling pathways and the ways they are coordinately controlled. In particular, the interaction of light and hormones in the control of cell elongation is not clear. Thus, there remains a need for the identification and characterization of additional mutants, and polypeptides encoded thereby, of enzymes involved in these pathways of plant growth.
Eukaryotic protein kinases are an extensive family of enzymes, many of which mediate the response of eukaryotic cells to external stimuli. One type of protein kinase, known as “SHAGGY,” is widespread in the plant kingdom and is a serine/threonine protein kinase which is homologous to the mammalian glycogen synthase kinase-3 (GSK-3). Plant homologs of GSK-3 have been found in such divergent plant species as Arabidopsis (Bianchi et al., Mol. Gen. Genet. (1994) 242:337-345; Jonak et al., Plant. Mol. Biol. (1995) 27:217-221; Dornelas et al., Plant Physiol. (1997) 113:306) Medicago (Pay et al., Plant J. (1993) 3:847-856), Nicotiana (Elinzenberger et al., Biochem. Biophys. Acta (1995) 1260:315-319); and Petunia (Decroocq-Ferrant et al., Plant J. (1995) 7:897-911), among others. Despite the widespread occurrence of SHAGGY protein kinases in higher plants, very little is known about the role these proteins play in plant development.